MicroRNA-124Suppresseses Glioblastoma Cell Proliferation And Invasive By Directly Targeting PPP1R13L

Background and AimsCancer is now the No.1cause of death for human in worldwide. Glioblastoma multiforme (GBM) is the the highest grade glima and is the most malignant form of astrocytoma which is designated as a grade IV glioma according to the World health organization (WHO)’s classification of the tumors of the central nervous system. Thus, Glioblastoma is among of the most common and lethal central nervous system neoplasm, with an annual incidence of4.96/100,000, and constituting about50%of all gliomas. Until now, various treatments maninly include surgery, radiation therapy, chemotherapy and combanatioin therapy can be used to treat glioblastoma. Despite great improvement for the treatment of glioblastoma have already been made in the past, however, the survival rate of patients with glioblastoma remains relatively low. As glioblastoma is usually localized in the cerebral white matter, grow quickly and highly invasive behavior, the life span of patients are extremely short, with a median survival time of approximately15months, and the median survival time of95%patients with glioblastoma from the time of diagnosis without any treatment is less than3months. Therefore, it is urgently needed to develop much more effective strategies and methods for diagnosis and treatment of glioblastoma. In past decades, lots of studies indicated that the carcinogenesis and progession of glioblastoma is a very complicated process that involving in the aberrant expression of oncogenes or tumor suppressor genes which lead to the activation or inactivation of signaling pathway in cells. Therefore, to further clarify the molecular mechanisms underlied the carcinogenesis and development of glioblastoma are especially important for diagnosis, prognosis and treatment of glioblastoma. A large progress for underling the molecular mechanisms of glioblastoma have been made before, however, the molecular mechanisms of glioblastoma are still largely unidentified.MicroRNAs are a class of small, non-coding, single-stranded RNAs. It was first identified in Caenorhabditis elegans in1993by Lee et al who found microRNA lin-4is required for the post-embryonic development for Caenorhabditis elegans. Since this discovery, miRNAs have been identified in diverse species including animals and plants. In humans, over400microRNA have been discovered and it is predicted that the human genome may encodes up to1000microRNAs. MicroRNAs can bind to the3’untranslated region (3’-UTR) of their target mRNAs for affecting mRNA stability at the transcriptional level, or bind to their mRNA target for blocking target gene expression at the level of protein translation. Accumulating data indicated that microRNAs are emerging as important regulators, involving in a serious of cellular events such as differentiation, proliferation, development, metastasis and apoptosis, etc. Due to the importance of microRNA for regulating various crucial cell process, the dysregulation of microRNA will no doubt change cellular differentiation, cell cycle control and apoptosis of normal cells, and finally lead to tumorigenesis. It is therefore that intervention of carcinogenesis and development of glioblastoma by microRNA will be a great promise therapeutic strategies in the future.microRNA-124is enriched in brain with a crucial role in neural development and has been shown to be down-regulated in glioma, suggesting it functions as a tumor suppressor in brain tumor progression. Studies showed that miR-124could regulate growth, invasiveness, stem-like traits, differentiation, apoptosis of glioblastoma cell and these processes were correlated to multiple target genes. Therefore, in present study, to further study the function of microRNA-124, we comparatively analyse the expression difference between glioblastoma tissue and normal brain tissue, and identified the potential target gene which plays key role in the carcinogenesis of glioblastoma by bioinformatics methods, and then examined the role of microRNA-124as well as it’s target gene PPP1R13L in regulation of differentiation, proliferation and invasive of glioblastoma. Our results may provide data for supporting microRNA- 124/PPP1R13L as novel therapeutic, diagnostic or prognostic tools for glioblastoma.Methods and Results1. MicroRNA-124is significantly down-regulated in glioblastoma tissues:To detect the expression of microRNA-124in glioblastoma tissues, we measured the expression level of microRNA-124in9cases of glioblastoma tissues and7cases of adjacent nontumor tissues using Real-Time quantitative RT-PCR. Our results showed that microRNA-124was markedly down-regulated in9examined samples compared with adjacent nontumor tissues (Normal) from the same patient, and the median decrease fold was3.5times, indicating that miR-124is significantly down-regulated in human glioblastoma.2. MicroRNA-124negatively regulates the expression of PPP1R13L gene:The potential microRNA binding sites on the3’-UTR of mRNA sequence of PPP1R13L gene were analysed with bioinformatics sofeware TargetScan. It was found that the3’-UTR of PPP1R13L mRNA contained two microRNA-124binding site, and one microRNA-124binding site was located at about180bp downstream from the3’-UTR of PPP1R13L mRNA, which is highly conserved across different species. To confirm whether PPP1R13L is a direct target of microRNA-124, we constructed wild-type3’-UTR of PPP1R13L mRNA as well as mutated3’-UTR of PPP1R13L mRNA (conservative microRNA-124binding site mutation) into the luciferase reporter plasmid. The lucifease assays indicated that over-expression of microRNA-124significantly inhibited the luciferase activity of wild-type construct, but have no effect on mutant construct. Using Real-Time PCR, we found that microRNA-124can significantly decrease the mRNA level of PPP1R13L in glioblastoma cells, U251cell and U373cell, respectively. Using Western blot, we further demonstrated that microRNA-124can markedly suppress the endogenous protein level of PPP1R13L in glioblastoma cells, U251cell and U373cell, respectively. Taken together, these results indicated that microRNA-124negatively regulates the expression of PPPlR13L gene.3. MicroRNA-124regulates proliferation of glioblastoma cell by targeting PPP1R13L. By using MTT and colony formation assays, we found that overexpression of microRNA-124dramatically decreased the growth rate of both glioblastoma cells (U251and U373) as compared with that of negative control (NC) transfected cells. However, inhibition of microRNA-124increased the growth rate of both glioma cells as compared with that of NC transfected cells. It suggests that microRNA-124regulates proliferation of glioblastoma cell by targeting PPP1R13L4. MicroRNA-124regulates Gl/S phase transition of glioblastoma cell by targeting PPP1R13L.By using flow cytometry, we found a significant increase in the percentage of cells in the G1/G0peak and a decrease in the percentage of cells in the S peak in the both microRNA-124overexpressing cells, and a decrease in G1/G0phase cells and an increase in S phase cells in the both glioma cells transfected with miR-124inhibitor. This results suggested that microRNA-124regulates G1/S phase transition of glioblastoma cell by targeting PPP1R13L.5. MicroRNA-124regulates invasions of glioblastoma cell by targeting PPP1R13L.By using transwell experiment, We observed that, compared to negative control (NC), overexpression of microRNA-124in U251and U373cells obviously inhibited their invasion, however, the invasion were increased when inhibition of microRNA-124in U251and U373cells. Meanwhile, we found that MMP-9and MMP-13as the molecular markers of cancer metastasis were decreased after transfected with miR-124mimics, and miR-124inhibitor increased their expression as compared with NC. This results indicated that microRNA-124regulates invasions of glioblastoma cell by targeting PPP1R13L.Main innovative Points1. For the first time, by using RT-PCR, the present study demonstrated that microRNA-124was decreased in glioblastoma tissue, which implicated microRNA-124as a potential diagnostic marker of glioblastoma.2. For the first time, we found the critical roles of microRNA-124in suppressing cells proliferation, cell cycle G1/S phase transition, invasion of glioblastoma cells by targeting PPP1R13L gene, which contribute to understande the molecular mechanism of carcinogenesis of glioblastoma, implicating that microRNA-124as a novel potential therapeutic target for glioblastoma.ConclusionsOur study demonstrated that microRNA-124was down-regulated in glioblastoma tissue which could be served as a diagonositc marker of glioblastoma, and documented the critical role of microRNA-124in inhibition of cell proliferation, cell cycle G1/S phase transition, invasion of glioblastoma cells by targeting PPP1R13L gene, which implicated that microRNA-124as a novel potential therapeutic target for glioblastoma.